Surrogate markers for viral infections and other inflammatory responses

ABSTRACT

Compositions and methods for the detection, diagnosis and treatment of BVDV and other viruses are provided.

This application claims priority to U.S. provisional Application No.60/757,965 filed Jan. 11, 2006, the entire contents of which areincorporated by reference herein.

Pursuant to 35 U.S.C. Section 202(c), it is acknowledged that the UnitedStates Government has certain rights in the invention described herein,which was made in part with funds from the USDA/CSREES, grant numbers2004-35204-14916 and 2004-35204-17005.

FIELD OF THE INVENTION

This invention relates to the field of molecular biology and virology.More specifically, the present invention provides materials and methodsfor the diagnosis and staging of bovine viral diarrhea virus (BVDV).

BACKGROUND OF THE INVENTION

Several publications and patent documents are cited throughout thisapplication in order to more fully describe the state of the art towhich this invention pertains. The disclosure of each of these citationsis incorporated by reference herein.

Bovine viral diarrhea virus (BVDV) costs the United States cattleindustry more than 400 million dollars per year. The pathogenesis ofBVDV infection has features that are unique to this virus and vary withthe time of infection, virulence of the viral strain, and age of theanimals at the time of infection.

When the infection occurs after 150 days of gestation (post-developmentof the immune system) or after birth, including adult animals, theinfection is referred to as acute infection. The clinical manifestationof acute infections with BVDV range from sub-clinical or unapparentinfections to embryonic death, abortions, stillborn, malformed or slowgrowing calves.

Certain strains of BVDV can cause a hemorrhagic syndrome with highmorbidity and moderate mortality in adult animals. Acutely infectedanimals usually recover and eliminate the virus within 10 to 14 dayspost infection.

Animals vaccinated with modified live vaccines against BVDV have animmune response similar to the one induced by natural, acute infection.In contrast, infection of the fetus during the first 150 days ofgestation, when the immune system has not yet developed, can lead to thegeneration of persistently infected (PI) calves. Some of these PI calvesdie soon after birth, but others live for relatively long periods oftime without showing any clinical signs. PI animals cannot eliminate theinfecting BVDV from their system, and continuously release high amountsof virus in their bodily secretions and excretions, making them acontinuous source of infection within the herd and potentially to otherherds as well. Furthermore, nursing PI calves can acutely infect theirmothers and other normal nursing calves, which in turn infect their ownmothers while they are pregnant, producing a new cycle of infection andeventually more PI calves.

Mucosal disease, an uncommon but fatal complication observed in PIcalves, occurs when the virus mutates or the animal is superinfectedwith an antigenically related BVDV virus. Current vaccines arerelatively inefficient in preventing fetal infections, therefore theidentification and elimination of PI animals is essential to anysuccessful program for control or eradication of BVDV.

Currently available tests for the detection of PI animals are based onthe identification of the viral antigen in a blood or tissue sample(most commonly a skin biopsy) using detection methods that depend on thespecific binding of anti-BVDV antibodies. Although these tests arewidely used for the detection of PI animals they frequently fail toidentify all infected animals (false negatives) resulting in the failureto remove all PI animals from the infected herd. Moreover, serologicaltests cannot differentiate between PIs and uninfected animals, orbetween acutely infected and vaccinated animals.

Identification and elimination of PI animals from an affected herd isthe most cost effective measure to control and eradicate BVDV,underscoring the criticality of an inexpensive and convenient diagnostictest. It is an object of the invention to provide such a test and kitfor performing the same.

SUMMARY OF THE INVENTION

In accordance with the present invention, methods and compositions fordiagnosis of Bovine Viral Diarrhea Virus (BVDV) are disclosed.Specifically, a simple, convenient test for accurately diagnosing BVDVis provided. The instant method provides the means to differentiatecattle persistently infected with BVDV (PI) from control non-infectedsteers. Other markers are provided which enable the skilled person toidentify 1) heifers carrying persistently infected fetuses; 2) heiferscarrying transiently virally infected fetuses and 3) heifers carryingcontrol, uninfected fetuses. Such differentiation may be accomplished bydetecting altered expression levels of one or more markers shown inTables 1-9, or the proteins or peptide fragments encoded thereby. Mostpreferably, the test can be easily conducted in the field byveterinarians or cattle producers.

In one aspect of the invention, BVDV surrogate markers are provided. ABVDV surrogate marker may be a nucleic acid or polypeptide or fragmentsthereof. Such markers are provided herein at Tables 1-9. Also providedin accordance with the invention are oligonucleotides, including probesand primers, that specifically hybridize with the nucleic acid sequencesset forth in Tables 1-9. Antibodies immunologically specific for theBVDV marker polypeptides described herein are also within the scope ofthe invention.

In a further aspect of the invention, recombinant DNA moleculescomprising the nucleic acid molecules set forth above, operably linkedto a vector are provided. The invention also encompasses host cellscomprising a vector encoding a BVDV specific marker of the invention.

In another aspect of the invention, methods for detecting adifferentially expressed BVDV specific marker molecules in a biologicalsample are provided. Such molecules can be BVDV specific marker nucleicacids, such as mRNA, DNA, cDNA, or BVDV specific marker polypeptides orfragments thereof. Preferably the BVDV surrogate marker exhibitsexpression levels which differ at least 2 fold from normal, uninfectedcattle. The BVDV markers of the invention may be up or down regulatedrelative to the levels observed in non-infected control cattle.Exemplary methods comprise detection of isolated biological moleculeswhich hybridize to BVDV specific markers which are affixed to a solidsupport, or mRNA analysis, for example by RT-PCR. Immunological methodsinclude for example contacting a sample with a detectably labeledantibody immunologically specific for a BVDV specific marker polypeptideand determining the presence of the polypeptide as a function of theamount of detectably labeled antibody bound by the sample relative tocontrol cells. In a preferred embodiment, these assays may be used todetect differentially expressed proteins encoded by the nucleic acidsset forth in Tables 1-9.

In a further aspect of the invention, kits for detection of BVDVinfection or lack thereof are provided. An exemplary kit comprises aBVDV specific marker protein, polynucleotide or a gene chip comprising aplurality of such polynucleotides, or antibody, which are optionallylinked to a detectable label. The kits may also include solid supports,pharmaceutically acceptable carriers and/or excipients, a suitablecontainer, and instructions for use.

In yet another aspect of the invention, the differentially regulatedBVDV markers described herein may be used in screening methods toidentify new therapeutic agents for the treatment of viral infections,including BVDV infection.

Agents which affect the differential expression of nucleic acids orproteins associated with BVDV infection may prove efficacious for thetreatment of BVDV.

BRIEF DESCRIPTION OF THE FIGURE

FIG. 1 is a schematic diagram showing some of the features of persistentand acute or transient BVDV infection.

FIG. 2 is a graph showing upregulation of interferon stimulated gene 15(ISG15) in blood from persistently infected calves. ISGIS mRNA levelswere determined using semi-quantitative (adjusted for GAPDH) Sybr greenReal Time PCR. Blood from three persistently infected (PI) and threecalves that had been vaccinated against BVDV (TI) are represented in theanalysis. ISG15 means differ between PI and TI (P<0.05).

FIG. 3 is a graph showing select blood cell markers that are upregulatedin bloods from persistently infected, when compared to non-infectedsteers using semi-quantitative Real Time PCR (GAPDH used as a control).The 28 kD (interferon induced 28 kD protein; CK771386), BST2 (bonemarrow stromal cell surface antigen 2; CK846889), MX2 (myxovirusresistance 2; NM_(—)173941) and ISG15 (Interferon stimulated 15kDa;NM_(—)174366) markers were all useful blood cell mRNA markers fordistinguishing persistent viral infection (positive) when compared tocontrol non-infected steers (negative). In this illustration, ISG15 andMX2 are preferred markers.

FIG. 4 is a heat plot illustrating three-Way ANOVA analysis of bloodcell gene expression from mothers carrying control vs. TI vs. PI virallyinfected fetuses described in Table 9. This analysis represents a foldchange of 1.5 fold or greater with P<0.01, Table 9 provides the actual Pvalues for each comparison and a more complete description of each gene.

DETAILED DESCRIPTION OF THE INVENTION

Bovine viral diarrhea virus (BVDV) provides a challenge to cattleproducers, because BVDV is a contagious and potentially lethal diseasethat is currently difficult and expensive to differentially diagnose.Current tests are performed on samples collected from young calves afterbirth. Thus, many of these infected calves have already shed virus andhave infected other pregnant cows. Therefore reinfection of pregnantcows helps maintain the infectious cycle.

The complex host-viral interactions resulting from persistent infectionare minimally understood, particularly in the bovine host. Thus, onepurpose of the present research was to identify those genes andassociated biological pathways which are activated or down-regulated inresponse to viral infection to facilitate a better understanding of themechanism of virus action. Another objective of the research was toidentify peripheral blood markers that will help distinguish pregnantcattle that are carrying persistently infected from those carryingtransiently virally infected fetuses. Depending on the time of infectionduring gestation, noncytopathic (ncp) bovine viral diarrhea virus (BVDV)causes persistent infection (PI, <150 d) or transient infection(TI, >150 d.) in fetuses. TI fetuses develop immunity to the viralstrain and clear the virus. PI fetuses do not recognize the virus as aforeign agent and once born continually shed the virus and infect othercattle. Detection and removal of pregnant cows or heifers carrying PIfetuses would greatly benefit the successful implementation of controlprograms. Provided herein is a simple and effective test for diagnosingBVDV, and identifying persistently infected (PI) animals.

Experimental evidence is provided which indicates that the pattern ofgene expression in vaccinated or acutely infected and PI animals isdifferent, and therefore the differential expression of genes can beused as a diagnostic marker for these types of BVDV infection. Genesthat are differentially expressed in the cells of the blood or the skinof persistently infected animals (surrogate markers) are identifiedusing gene chip analysis of mRNA of PI when compared to vaccinated oracutely infected animals. Antibodies produced against such surrogatemarkers can be used to develop a diagnostic test to detect PI animals,by analyzing the presence of the surrogate marker in an animal's bloodor skin.

Thus, in accordance with the present invention, gene chip analysis hasbeen performed on nucleic acids obtained from blood cells collected frombovines that are persistently infected with BVDV when compared tovaccinated control bovines.

In yet another aspect, the differentially regulated BVDV markersdescribed herein may be used in screening methods to identify newtherapeutic agents for the treatment of viral infections, particularlyBVDV infections. For example, agents which down regulate the expressionof genes which are upregulated in response to infection may haveefficacy as antiviral agents.

I. Definitions

The following definitions are provided to facilitate an understanding ofthe present invention: The term “surrogate marker” or infection markeris a marker which is differentially expressed in animals infected with apathological condition, such as a virus.

Specifically, a surrogate marker may be any gene expression productwhich is differentially expressed in persistently infected animals whencompared to vaccinated or acutely infected animals, transiently infectedand non-infected or non-vaccinated (normal) animals. A surrogate markercan be a polynucleotide, a protein, a peptide, or any gene expressionproduct, but is preferably an mRNA or protein expression product. Thesurrogate markers described herein may also be useful for diagnosinginvention with other RNA viruses which include for example, Influenza,HIV, Ebola virus, FeLv, FIP virus, Bluetongue virus, West Nile Virus,hepatitis C Virus and Epizootic Hemorrhagic Disease Virus. Thus, theterm “surrogate marker” as used herein refers to those biologicalmolecules which are differentially expressed in response to infectionwith any RNA virus.

A persistently infected calf is one that is infected in utero prior to150 days of gestation, does not clear the virus and if it survives willcontinue to shed virus. A transiently infected calf is one that isinfected in utero after 150 days of gestation, recovers and clears thevirus. An acutely infected animal is one that is infected postnatallyand recovers, clearing the virus, A control animal is one that was neverinfected with virus.

A “BVDV surrogate marker” refers to a marker which is differentiallyexpressed in animals infected with BVDV. Specifically, a BVDV surrogatemarker may be any gene expression product which is differentiallyexpressed in any or all of acutely infected BVDV animals, persistentlyinfected BVDV animals, vaccinated BVDV animals, and normal animals. Asurrogate marker can be a polynucleotide, a protein or peptide, or anygene expression product, but is preferably an mRNA or protein expressionproduct.

A “BVDV surrogate marker profile” is an expression pattern of surrogateBVDV markers which correlates specifically to acute BVDV infection,persistent BVDV infection, BVDV vaccinated cattle, or non-BVDV infectedcattle.

A “sample” or “patient sample” or “biological sample” generally refersto a sample which may be tested for a particular molecule, preferably asurrogate BVDV marker, including one or more surrogate BVDVpolynucleotide, polypeptide, or antibody. Samples may include but arenot limited to blood or skin, serum, plasma, urine, saliva, and thelike. Most preferably, the sample is a skin sample or a blood samplefrom cattle.

“Blood” includes but is not limited to whole blood, blood treated ormixed with anticoagulants, and any component of whole blood, includingbut not limited to serum, plasma, buffy coat, and purified peripheralblood mononuclear cells.

A “ruminant” is an even-toed, herbivorous, ungulate mammal (OrderArtiodactyla) that chews cud (ruminate) and has a complex, usuallyfour-chambered stomach containing micro-organisms that break downcellulose. Ruminants include but are not limited to cattle, sheep,antelope, deer, giraffes, elk, moose, caribou, yak and camelids (e.g.,camel, llama, alpaca, vicuna and guanaco).

The term “cattle” as used herein includes any of numerous types ofdomestic quadrupeds held as property or raised for use, such aslivestock, cows, bulls, bovine, steer, oxen, bison, and the like. Theterm “cattle” generally refers to multiple animals, but may alsodescribe a single animal.

The term “ruminant nucleic acid” or “ruminant protein” refers to anucleic acid or protein whose sequence is of ruminant origin.Preferably, a ruminant nucleic acid or ruminant protein is of bovineorigin.

A “BVDV surrogate marker detector molecule” is a molecule whichfacilitates detecting or quantitating a BVDV surrogate marker. A BVDVsurrogate marker detector molecule can be any molecule which facilitatesdetection of BVDV surrogate marker, including but not limited to a probeor primer which specifically hybridizes with a BVDV surrogate markernucleic acid, or an antibody or fragment thereof which specificallybinds to a BVDV surrogate marker polypeptide or peptide fragment.

The term “differential diagnosis” refers to a diagnosis which is able todifferentiate between two or more different types of BVDV infection (forexample, acute infection, persistent infection, or not infected.) Thistest also identifies previously vaccinated animals.

The phrase “consisting essentially of” when referring to a particularnucleotide or amino acid means a sequence having the properties of agiven SEQ ID NO:.

For example, when used in reference to an amino acid sequence, thephrase includes the sequence per se and molecular modifications thatwould not affect the functional and unique characteristics of thesequence.

The term “nucleic acid molecule” describes a polymer ofdeoxyribonucleotides (DNA) or ribonucleotides (RNA). The nucleic acidmolecule may be isolated from a natural source by cDNA cloning orsubtractive hybridization or synthesized manually. The nucleic acidmolecule may be synthesized manually by the triester synthetic method orby using an automated DNA synthesizer.

With regard to nucleic acids used in the invention, the term “isolatednucleic acid” is sometimes employed. This term, when applied to DNA,refers to a DNA molecule that is separated from sequences with which itis immediately contiguous (in the 5′ and 3′ directions) in the naturallyoccurring genome of the organism or virus from which it was derived. Forexample, the “isolated nucleic acid” may comprise a DNA moleculeinserted into a vector, such as a plasmid or virus vector, or integratedinto the genomic DNA of a prokaryote or eukaryote cells.

An “isolated nucleic acid molecule” may also comprise a eDNA molecule.An isolated nucleic acid molecule inserted into a vector is alsosometimes referred to herein as a recombinant nucleic acid molecule.

The term “complementary” describes two nucleotides that can formmultiple favorable interactions with one another. For example, adenineis complementary to thymine as they can form two hydrogen bonds.Similarly, guanine and cytosine are complementary since they can formthree hydrogen bonds. Thus if a nucleic acid sequence contains thefollowing sequence of bases, thymine, adenine, guanine and cytosine, a“complement” of this nucleic acid molecule would be a moleculecontaining adenine in the place of thymine, thymine in the place ofadenine, cytosine in the place of guanine, and guanine in the place ofcytosine. Because the complement can contain a nucleic acid sequencethat forms optimal interactions with the parent nucleic acid molecule,such a complement can bind with high affinity to its parent molecule.

With respect to single stranded nucleic acids, particularlyoligonucleotides, the term “specifically hybridizing” refers to theassociation between two single-stranded nucleotide molecules ofsufficiently complementary sequence to permit such hybridization underpre-determined conditions generally used in the art (sometimes termed“substantially complementary”). In particular, the term refers tohybridization of an oligonucleotide with a substantially complementarysequence contained within a single-stranded DNA or RNA molecule of theinvention, to the substantial exclusion of hybridization of theoligonucleotide with single- stranded nucleic acids of non-complementarysequence. Appropriate conditions enabling specific hybridization ofsingle stranded nucleic acid molecules of varying complementarity arewell known in the art.

For instance, one common formula for calculating the stringencyconditions required to achieve hybridization between nucleic acidmolecules of a specified sequence homology is set forth below (Sambrooket al. , Molecular Cloning, Cold Spring Harbor Laboratory (1989):

Tm 81.5° C +16.6 Log [Na+]+0.41 (% G+C)−0.63 (% formamide)−600/#bp induplex.

As an illustration of the above formula, using [Na+]=[0.368] and 50%formamide, with GC content of 42% and an average probe size of 200bases, the Tm is 57° C. The Tm of a DNA duplex decreases by 1-1.5° C.with every 1% decrease in homology. Thus, targets with greater thanabout 75% sequence identity would be observed using a hybridizationtemperature of 42° C.

The stringency of the hybridization and wash depend primarily on thesalt concentration and temperature of the solutions. In general, tomaximize the rate of annealing of the probe with its target, thehybridization is usually carried out at salt and temperature conditionsthat are 20-25° C. below the calculated Tm of the hybrid.

Wash conditions should be as stringent as possible for the degree ofidentity of the probe for the target. In general, wash conditions areselected to be approximately 12-20° C. below the Tm of the hybrid. Inregards to the nucleic acids of the current invention, a moderatestringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 pg/ml denatured salmon sperm DNAat 42° C., and washed in 2×SSC and 0.5% SDS at 55° C. for 15 minutes. Ahigh stringency hybridization is defined as hybridization in 6×SSC, 5×Denhardt's solution, 0.5% SDS and 100 pg/ml denatured salmon sperm DNAat 42° C., and washed in 1×SSC and 0.5% SDS at 65° C. for 15 minutes. Avery high stringency hybridization is defined as hybridization in 6×SSC,5× Denhardt's solution, 0.5% SDS and 100 pg/ml denatured salmon spermDNA at 42° C., and washed in 0.1×SSC and 0.5% SDS at 65° C. for 15minutes.

The term “oligonucleotide” as used herein refers to primers and probesof the present invention, and is defined as a nucleic acid moleculecomprised of two or more ribo-or deoxyribonucleotides, preferably morethan three. The exact size of the oligonucleotide will depend on variousfactors and on the particular application and use of theoligonucleotide. Oligonucleotides, which include probes and primers, canbe any length from 3 nucleotides to the full length of the nucleic acidmolecule, and explicitly include every possible number of contiguousnucleic acids from 3 through the full length of the polynucleotide.Preferably, oligonucleotides, which include probes and/or primers are atleast about 10 nucleotides in length, more preferably at least 15nucleotides in length, more preferably at least about 20 nucleotides inlength.

The term “probe” as used herein refers to an oligonucleotide,polynucleotide or nucleic acid, either RNA or DNA, whether occurringnaturally as in a purified restriction enzyme digest or producedsynthetically, which is capable of annealing with or specificallyhybridizing to a nucleic acid with sequences complementary to the probe.A probe may be either single-stranded or double-stranded. The exactlength of the probe will depend upon many factors, includingtemperature, source of probe and use of the method. For example, fordiagnostic applications, depending on the complexity of the targetsequence, the oligonucleotide probe typically contains 15-25 or morenucleotides, although it may contain fewer nucleotides. The probesherein are selected to be complementary to different strands of aparticular target nucleic acid sequence.

This means that the probes must be sufficiently complementary so as tobe able to “specifically hybridize” or anneal with their respectivetarget strands under a set of pre-determined conditions. Therefore, theprobe sequence need not reflect the exact complementary sequence of thetarget. For example, a non-complementary nucleotide fragment may beattached to the 5′ or 3′end of the probe, with the remainder of theprobe sequence being complementary to the target strand. Alternatively,non-complementary bases or longer sequences can be interspersed into theprobe, provided that the probe sequence has sufficient complementaritywith the sequence of the target nucleic acid to anneal therewithspecifically.

The term “primer” as used herein refers to an oligonucleotide, eitherRNA or DNA, either single- stranded or double-stranded, either derivedfrom a biological system, generated by restriction enzyme digestion, orproduced synthetically which, when placed in the proper environment, isable to functionally act as an initiator of template-dependent nucleicacid synthesis. When presented with an appropriate nucleic acidtemplate, suitable nucleoside triphosphate precursors of nucleic acids,a polymerase enzyme, suitable cofactors and conditions such as asuitable temperature and pH, the primer may be extended at its 3terminus by the addition of nucleotides by the action of a polymerase orsimilar activity to yield a primer extension product. The primer mayvary in length depending on the particular conditions and requirement ofthe application. For example, in diagnostic applications, theoligonucleotide primer is typically 15-25 or more nucleotides in length.The primer must be of sufficient complementarity to the desired templateto prime the synthesis of the desired extension product, that is, to beable anneal with the desired template strand in a manner sufficient toprovide the 3 ′hydroxyl moiety of the primer in appropriatejuxtaposition for use in the initiation of synthesis by a polymerase orsimilar enzyme. It is not required that the primer sequence represent anexact complement of the desired template.

For example, a non-complementary nucleotide sequence may be attached tothe 5′end of an otherwise complementary primer. Alternatively,non-complementary bases may be interspersed within the oligonucleotideprimer sequence, provided that the primer sequence has sufficientcomplementarity with the sequence of the desired template strand tofunctionally provide a template-primer complex for the synthesis of theextension product. Polymerase chain reaction (PCR) has been described inU.S. Pat. Nos. 4,683,195, 4,800,195, and 4,965,188, the entiredisclosures of which are incorporated by reference herein.

The term “vector” relates to a single or double stranded circularnucleic acid molecule that can be transfected or transformed into cellsand replicate independently or within the host cell genome. A circulardouble stranded nucleic acid molecule can be cut and thereby linearizedupon treatment with restriction enzymes. An assortment of vectors,restriction enzymes, and the knowledge of the nucleotide sequences thatare targeted by restriction enzymes are readily available to thoseskilled in the art. A vector of the invention includes any replicon,such as a plasmid, cosmid, bacmid, phage or virus, to which anothergenetic sequence or element (either DNA or RNA) may be attached so as tobring about the replication of the attached sequence or element. Anucleic acid molecule of the invention can be inserted into a vector bycutting the vector with restriction enzymes and ligating the two piecestogether.

Many techniques are available to those skilled in the art to facilitatetransformation, transfection, or transduction of the expressionconstruct into a prokaryotic or eukaryotic organism. The terms“transformation”, “transfection”, and “transduction” refer to methods ofinserting a nucleic acid and/or expression construct into a cell or hostorganism. These methods involve a variety of techniques, such astreating the cells with high concentrations of salt, an electric field,or detergent, to render the host cell outer membrane or wall permeableto nucleic acid molecules of interest, microinjection, PEG-fusion, andthe like.

The term “promoter element” describes a nucleotide sequence that isincorporated into a vector that, once inside an appropriate cell, canfacilitate transcription factor and/or polymerase binding and subsequenttranscription of portions of the vector DNA into mRNA. In oneembodiment, the promoter element of the present invention precedes theSend of the BVDV surrogate marker nucleic acid molecule such that thelatter is transcribed into mRNA. Host cell machinery then translatesmRNA into a polypeptide.

Those skilled in the art will recognize that a nucleic acid vector cancontain nucleic acid elements other than the promoter element and theBVDV surrogate marker gene nucleic acid molecule. These other nucleicacid elements include, but are not limited to, origins of replication,ribosomal binding sites, nucleic acid sequences encoding drug resistanceenzymes or amino acid metabolic enzymes, and nucleic acid sequencesencoding secretion signals, periplasm or peroxisome localizationsignals, or signals useful for polypeptide purification.

An “expression operon” refers to a nucleic acid segment that may possesstranscriptional and translational control sequences, such as promoters,enhancers, translational start signals (e.g., ATG or AUG codons),polyadenylation signals, terminators, and the like, and which facilitatethe expression of a polypeptide coding sequence in a host cell ororganism.

As used herein, the terms “reporter”, “reporter system”, “reportergene”, or “reporter gene product” shall mean an operative genetic systemin which a nucleic acid comprises a gene that encodes a product thatwhen expressed produces a reporter signal that is a readily measurable,e.g., by biological assay, immunoassay, radio immunoassay, or bycalorimetric, fluorogenic, chemiluminescent or other methods. Thenucleic acid may be either RNA or DNA, linear or circular, single ordouble stranded, antisense or sense polarity, and is operatively linkedto the necessary control elements for the expression of the reportergene product. The required control elements will vary according to thenature of the reporter system and whether the reporter gene is in theform of DNA or RNA, but may include, but not be limited to, suchelements as promoters, enhancers, translational control sequences, polyA addition signals, transcriptional termination signals and the like.

The introduced nucleic acid may or may not be integrated (covalentlylinked) into nucleic acid of the recipient cell or organism. Inbacterial, yeast, plant and mammalian cells, for example, the introducednucleic acid may be maintained as an episomal element or independentreplicon such as a plasmid. Alternatively, the introduced nucleic acidmay become integrated into the nucleic acid of the recipient cell ororganism and be stably maintained in that cell or organism and furtherpassed on or inherited to progeny cells or organisms of the recipientcell or organism. Finally, the introduced nucleic acid may exist in therecipient cell or host organism only transiently.

The term “selectable marker gene” refers to a gene that when expressedconfers a selectable phenotype, such as antibiotic resistance, on atransformed cell.

The term “operably linked” means that the regulatory sequences necessaryfor expression of the coding sequence are placed in the DNA molecule inthe appropriate positions relative to the coding sequence so as toeffect expression of the coding sequence. This same definition issometimes applied to the arrangement of transcription units and othertranscription control elements (e.g., enhancers) in an expressionvector.

The term “tag”, “tag sequence”, or “protein tag” refers to a chemicalmoiety, either a nucleotide, oligonucleotide, polynucleotide or an aminoacid, peptide or protein or other chemical, that when added to anothersequence, provides additional utility or confers useful properties,particularly in the detection or isolation, of that sequence. Thus, forexample, a homopolymer nucleic acid sequence or a nucleic acid sequencecomplementary to a capture oligonucleotide may be added to a primer orprobe sequence to facilitate the subsequent isolation of an extensionproduct or hybridized product. In the case of protein tags, histidineresidues (e. g., 4 to 8 consecutive histidine residues) may be added toeither the amino-or carboxy-terminus of a protein to facilitate proteinisolation by chelating metal chromatography. Alternatively, amino acidsequences, peptides, proteins or fusion partners representing epitopesor binding determinants reactive with specific antibody molecules orother molecules (e. g., flag epitope, c-mye epitope, transmembraneepitope of the influenza A virus hemaglutinin protein, protein A,cellulose binding domain, calmodulin binding protein, maltose bindingprotein, chitin binding domain, glutathione S-transferase, and the like)may be added to proteins to facilitate protein isolation by proceduressuch as affinity or immunoaffinity chromatography.

Chemical tag moieties include such molecules as biotin, which may beadded to either nucleic acids or proteins and facilitates isolation ordetection by interaction with avidin reagents, and the like. Numerousother tag moieties are known to, and can be envisioned by the trainedartisan, and are contemplated to be within the scope of this definition.

A “specific binding pair” comprises a specific binding member (sbm) anda binding partner (bp) which have a particular specificity for eachother and which in normal conditions bind to each other in preference toother molecules. Examples of specific binding pairs are antigens andantibodies, ligands and receptors and complementary nucleotidesequences. The skilled person is aware of many other examples. Further,the term “specific binding pair” is also applicable where either or bothof the specific binding member and the binding partner comprise a partof a large molecule. In embodiments in which the specific binding paircomprises nucleic acid sequences, they will be of a length to hybridizeto each other under conditions of the assay, preferably greater than 10nucleotides long, more preferably greater than 15 or 20 nucleotideslong.

An “antibody” or “antibody molecule” is any immunoglobulin, includingantibodies and fragments thereof that binds to a specific antigen. Theterm includes polyclonal, monoclonal, chimeric, and bispecificantibodies. Exemplary antibody fragments, capable of binding an antigenor other binding partner, are Fab fragment consisting of the VL, VH, Cland CH1 domains; the Fd fragment consisting of the VH and CH1 domains;the Fv fragment consisting of the VL and VH domains of a single arm ofan antibody; the dAb fragment which consists of a VH domain; isolatedCDR regions and F (ab′) 2 fragments, a bivalent fragment including twoFab fragments linked by a disulphide bridge at the hinge region. Singlechain Fv fragments are also included.

With respect to antibodies, the term “immunologically specific” refersto antibodies that bind to one or more epitopes of a protein or compoundof interest, but which do not substantially recognize and bind othermolecules in a sample containing a mixed population of antigenicbiological molecules. Exemplary antibodies bind to a protein or peptidefragment encoded by a nucleotide sequence set forth in Tables 1-9.

A “detection reagent” or a “marker detection reagent” is any substancewhich has binding affinity for a BVDV specific molecule, and includesbut is not limited to nucleic acid molecules with sufficient affinity tohybridize to the BVDV specific marker, probes, primers, antibodies,fragments thereof and the like. The “detection reagent” or “markerdetection reagent” may optionally be detectably labeled.

The term “detectable label” is used herein to refer to any substancewhose detection or measurement, either directly or indirectly, byphysical or chemical means, is indicative of the presence of a targetbioentity in a test sample. Representative examples of useful detectablelabels, include, but are not limited to the following: molecules or ionsdirectly or indirectly detectable based on light absorbance,fluorescence, reflectance, light scatter, phosphorescence, orluminescence properties; molecules or ions detectable by theirradioactive properties; molecules or ions detectable by their nuclearmagnetic resonance or paramagnetic properties. Included among the groupof molecules indirectly detectable based on light absorbance orfluorescence, for example, are various enzymes which cause appropriatesubstrates to convert, e. g., from non-light absorbing to lightabsorbing molecules, or from non-fluorescent to fluorescent molecules.

II. Surrogate BVDV Nucleic Acid Molecules, Probes, and Primers andMethods of Preparing the Same

Encompassed by the invention are surrogate BVDV nucleic acid molecules,nucleic acid molecules which encode isolated, enriched, or purifiedsurrogate BVDV proteins or peptides, including allelic variations,analogues, fragments, derivatives, mutants, and modifications of thesame.

Surrogate BVDV nucleic acid molecules, and nucleic acid sequencesencoding surrogate BVDV proteins may be isolated from appropriatebiological sources using methods known in the art. In a preferredembodiment, a cDNA clone is isolated from a cDNA expression library ofbovine origin. Preferably, the sample is isolated from a bovine whichhas been vaccinated for, or has acute, or persistent BVDV infection,Surrogate BVDV marker polynucleotides can be any one of, or anycombination of the markers shown in Tables 1-9, and further may includevariants which are at least about 75%, or 80% or 85% or 90% or 95%, andoften, more than 90%, or more than 95% homologous to the markers shownin Tables 1-9, over the full length sequence. Surrogate BVDV markerpolynucleotides also may be 60% or 65% or 70% or 75% or 80% or 85% or90% or 95% or 97% or 98% or 99% or greater than 99% homologous to themarkers shown in Tables 1-9, over the full length sequence. All homologymay be computed by algorithms known in the art, such as BLAST, describedin Altschul et al. (1990), J. Mol. Biol. 215: 403-10, or theSmith-Waterman homology search algorithm as implemented in MPSRCHprogram (Oxford Molecular). Someone of ordinary skill in the art wouldreadily be able to determine the ideal gap open penalty and gapextension penalty for a particular nucleic acid sequence.

Exemplary search parameters for use with the MPSRCH program in order toidentify sequences of a desired sequence identity are as follows: gapopen penalty:-16; and gap extension penalty:-4.

Degenerate variants are also encompassed by the instant invention. Thedegeneracy of the genetic code permits substitution of certain codons byother codons, which specify the same amino acid and hence would giverise to the same protein. The nucleic acid sequence can varysubstantially since, with the exception of methionine and tryptophan,the known amino acids can be coded for by more than one codon. Thus,portions or all of the markers could be synthesized to give a nucleicacid sequence significantly different from that shown in Tables 1-9. Theencoded amino acid sequence thereof would, however, be preserved.

In addition, the nucleic acid sequence may comprise a nucleotidesequence which results from the addition, deletion or substitution of atleast one nucleotide to the 5′-end and/or the 3′-end of one or more ofthe markers shown in Tables 1-9, or a derivative thereof. Any nucleotideor polynucleotide may be used in this regard, provided that itsaddition, deletion or substitution does not alter the amino acidsequence which is encoded by the nucleotide sequence, or it still sharesa region of homology with one or more of the markers shown in Tables1-9. For example, the present invention is intended to include anynucleic acid sequence resulting from the addition of ATG as aninitiation codon at the 5′-end of the surrogate BVDV marker nucleic acidsequence or its functional derivative, or from the addition of TTA, TAGor TGA as a termination codon at the 3′-end of the inventive nucleotidesequence or its derivative. Moreover, the nucleic acid molecule of thepresent invention may, as necessary, have restriction endonucleaserecognition sites added to its 5′-end and/or 3′-end.

Such functional alterations of a given nucleic acid sequence afford anopportunity to promote secretion and/or processing of heterologousproteins encoded by foreign nucleic acid sequences fused thereto. Allvariations of the nucleotide sequence of the markers shown in Tables 1-9and fragments thereof permitted by the genetic code are, therefore,included in this invention.

In an alternative embodiment, utilizing the sequence informationprovided by the eDNA sequence, genomic clones encoding a surrogate BVDVmarker gene may be isolated.

Alternatively, eDNA or genomic clones having homology with the markersshown in Tables 1-9 may be isolated from other species, such as mouse orhuman, using oligonucleotide probes corresponding to predeterminedsequences within surrogate BVDV marker gene.

III. Surrogate BVDV Proteins (Antigens) and Methods of Making the Same

Encompassed by the invention are isolated, purified, or enrichedsurrogate BVDV polypeptides, including allelic variations, analogues,fragments, derivatives, mutants, and modifications of the same which aredifferentially expressed in BVDV animals. Preferably, surrogate BVDVmarker polypeptides include polypeptides encoded by one or more of thesequences shown in Tables 1-9. Surrogate BVDV marker function is definedabove, and includes increased or decreased expression in response toBVDV infection, cross-reactivity with an antibody reactive with thepolypeptides encoded by one or more of the sequences shown in Tables 1-9or sharing an epitope with the same (as determined for example byimmunological cross-reactivity between the two polypeptides). SurrogateBVDV marker polypeptides or proteins can be encoded by one or more ofthe sequences shown in Tables 1-9, and further may include variantswhich are at least about 75%, or 80% or 85% or 90% or 95%, and often,more than 90%, or more than 95% homologous to the same over the fulllength sequence.

Surrogate BVDV marker polypeptides also may be 60% or 65% or 70% or 75%or 80% or 85% or 90% or 95% or 97% or 98% or 99% or greater than 99%homologous to polypeptides encoded by one or more of the sequences shownin Tables 1-9 over the full length sequence. All homology may becomputed by algorithms known in the art, such as BLAST, described inAltschul et al. (1990), J. Mol. Biol. 215: 403-10, or the Smith-Watermanhomology search algorithm as implemented in MPSRCH program (OxfordMolecular). Someone of ordinary skill in the art would readily be ableto determine the ideal gap open penalty and gap extension penalty for aparticular protein sequence. Exemplary search parameters for use withthe MPSRCH program in order to identify sequences of a desired sequenceidentity are as follows: gap open penalty:-12 ; and gap extensionpenalty:-2.

A full-length or truncated surrogate BVDV protein of the presentinvention may be prepared in a variety of ways, according to knownmethods. The protein may be purified from appropriate sources, e.g.,transformed bacterial or animal cultured cells or tissues, byimmunoaffinity purification. Additionally, the surrogate BVDV proteinmay be produced using in vitro expression methods known in the art. Forexample, a cDNA or gene may be cloned into an appropriate in vitrotranscription vector, such as pSP64 or pSP65 for in vitro transcription,followed by cell-free translation in a suitable cell-free translationsystem, such as wheat germ or rabbit reticulocyte lysates. In vitrotranscription and translation systems are commercially available, e. g.,from Promega Corp., Madison, Wis. or Invitrogen Corp., Carlsbad, Calif.

The surrogate BVDV proteins produced by gene expression in a recombinantprokaryotic or eukaryotic system may be purified according to methodsknown in the art. In a preferred embodiment, a commercially availableexpression/secretion system can be used, whereby the recombinant proteinis expressed and thereafter secreted from the host cell, to be easilypurified from the surrounding medium. If expression/secretion vectorsare not used, an alternative approach involves purifying the recombinantprotein by affinity separation, such as by immunological interactionwith antibodies that bind specifically to the recombinant protein ornickel columns for isolation of recombinant proteins tagged with 6-8histidine residues at their N-terminus or C-terminus.

Alternative tags may comprise the FLAG epitope or the hemagglutininepitope. Such methods are commonly used by skilled practitioners.

IV. Anti-Surrogate BVDV Protein Antibodies and Methods of Making theSame

The present invention also provides methods of making and usingantibodies capable of immunospecifically binding to surrogate BVDVproteins. Polyclonal antibodies directed toward surrogate BVDV proteinsmay be prepared according to standard methods. In a preferredembodiment, monoclonal antibodies are prepared, which reactimmunospecifically with the various epitopes on the surface of thesurrogate BVDV protein. Monoclonal antibodies may be prepared accordingto general methods of Kohler and Milstein, following standard protocols.

Purified BVDV antigens, or fragments thereof, may be used to producepolyclonal or monoclonal antibodies which also may serve as sensitivedetection reagents for the various types of BVDV infection (acute, PI,vaccination reaction, and not infected). Recombinant techniques enableexpression of fusion proteins containing part or all of BVDV. Thesurrogate BVDV protein itself, or surface proteins or antigens from thesurrogate BVDV protein may be used to advantage to generate an array ofmonoclonal antibodies specific for various epitopes of the surrogateBVDV protein, thereby providing even greater sensitivity for detectionof the surrogate BVDV protein (and thus BVDV infection) in samples.

Polyclonal or monoclonal antibodies that immunospecifically interactwith BVDV antigens can be utilized for identifying and diagnosing BVDV.For example, antibodies may be utilized for affinity separation ofproteins with which they immunospecifically interact. Antibodies mayalso be used to immunuoprecipitate proteins from a sample containing amixture of proteins and other biological molecules.

Other uses of anti-surrogate BVDV protein antibodies are describedbelow.

V. Methods of Using Surrogate BVDV Polynucleotides, Polypeptides, andAntibodies for Screening and Diagnostic Assays

Surrogate BVDV nucleic acids may be used for a variety of purposes inaccordance with the present invention. Surrogate BVDV nucleic acids(DNA, RNA, fragments thereof, etc.), or protein-encoding DNA, RNA, orfragments thereof may be used as probes to detect the presence ofsurrogate BVDV nucleic acids or protein in a sample. Methods in whichsurrogate BVDV nucleic acids and protein-encoding nucleic acids may beutilized as probes for such assays include, but are not limited to, (1)in situ hybridization; (2) Southern hybridization (3) northernhybridization; (4) gene chip analysis and (5) assorted amplificationreactions such as polymerase chain reactions (PCR).

Exemplary surrogate BVDV nucleic acids and nucleic acids encodingexemplary surrogate BVDV proteins or peptides are described in Tables1-9.

The surrogate BVDV nucleic acids of the invention may also be utilizedas probes to identify related surrogate BVDV variants. As is well knownin the art, hybridization stringencies may be adjusted to allowhybridization of nucleic acid probes with complementary sequences ofvarying degrees of homology. Thus, BVDV surrogate marker nucleic acidsmay be used to advantage to identify and characterize other genes ofvarying degrees of relation to BVDV surrogate markers, thereby enablingfurther characterization of BVDV surrogate markers. Additionally, theymay be used to identify genes encoding proteins that interact with BVDVsurrogate markers (e. g., by the “interaction trap” technique-see forexample Current Protocols in Molecular Biology, ed. Ausubel, F. M., etal., John Wiley & Sons, NY, 1997), which should further accelerateidentification of the molecular components involved in BVDV. Finally,they may be used in assay methods to detect BVDV.

Polyclonal or monoclonal antibodies immunologically specific forproteins encoded by BVDV surrogate markers or peptide fragments thereofmay be used in a variety of assays designed to detect and quantitate theprotein, as well as to detect ruminant BVDV by detecting upregulation ofBVDV surrogate markers. Such assays include, but are not limited to: (1)flow cytometric analysis; (2) immunochemical localization of BVDVspecific markers in a body cell, tissue, or fluid; and (3) immunoblotanalysis (e. g., dot blot, Western blot) (4) ELISA; (5) radioimmunoassayof extracts from various cells.

Additionally, as described above, anti-surrogate BVDV marker protein canbe used for purification of surrogate BVDV markers (e. g., affinitycolumn purification, immunoprecipitation).

Further, assays for detecting and quantitating surrogate BVDV markers,or to detect ruminant BVDV by detecting upregulation of BVDV specificmarkers may be conducted on any type of biological sample whereupregulation of these molecules is observed, including but not limitedto body fluids (including blood, serum, plasma, milk, or saliva), anytype of cell (such as skin cells, or blood cells, or endothelial cells),or body tissue.

From the foregoing discussion, it can be seen that surrogate BVDV markernucleic acids, surrogate BVDV marker expressing vectors, surrogate BVDVmarker proteins and anti-surrogate BVDV marker antibodies of theinvention can be used to detect surrogate BVDV marker expression in bodytissue, cells, or fluid, and alter BVDV specific marker proteinexpression for purposes of assessing the genetic and proteininteractions involved in BVDV and infection.

In most embodiments for screening for surrogate BVDV mRNA, surrogateBVDV nucleic acid in the sample will initially be amplified, e. g. usingPCR, to increase the amount of the template as compared to othersequences present in the sample. This allows the target sequences to bedetected with a high degree of sensitivity if they are present in thesample.

Thus any of the aforementioned techniques may be used as a diagnostictool for detecting surrogate BVDV markers.

Further, these techniques could be used to diagnose infectious diseasesin humans, by detection of a surrogate marker (rather than a viralantigen). For example, differential gene expression could be measured inHIV, Ebola, Hepatitis, and Herpes viral infections, etc. These tests areadvantageous in that they are directed to detection of a theoreticallyharmless surrogate marker, rather than the infectious agent itself.

Such techniques could also be used to diagnose infectious diseases incompanion animals by detection of a surrogate marker (rather than aviral antigen). An example of a potential application would be thediagnosis of feline infectious peritonitis of cats and latent viralinfections caused by herpes viruses for which current diagnostic tests(based on isolation and characterization of the virus) have a marginalreliability. In addition, this technology could also be used for thediagnosis of cancer through the identification of surrogate cancermarkers.

The instant inventive method improves upon the accuracy of current BVDVtests. A combination test, which measures both BVDV itself, and also oneor more BVDV surrogate marker, to differentially diagnose BVDV infectionprovides superior diagnostic results in the field.

VI. Assays for Differentially Diagnosing BVDV Using Specific SurrogateMarkers

In accordance with the present invention, it has been discovered thatBovine Viral Diarrhea Virus (BVDV) is correlated with increasedexpression levels of certain markers, including but not limited to mRNAsand proteins.

Thus, these molecules may be utilized in conventional assays todifferentially diagnose BVDV. The detection of one or more of thesedifferentially expressed BVDV surrogate molecules in a sample isindicative of BVDV. Similarly, specific patterns of expression allowdetection of acute versus persistent infection. Alternatively, theabsence of these molecules in a sample indicates that a ruminant is notinfected with BVDV.

In an exemplary method, a blood sample is obtained from a bovinesuspected of having an acute or persistent BVDV infection. Optionally,the blood may be centrifuged through a Hypaque gradient to obtain thebuffy coat. The blood or buffy coat preparation is diluted and subjectedto polymerase chain reaction conditions suitable for amplification ofthe BVDV surrogate marker encoding mRNA.

In certain applications, it may be necessary to include an agent, whichlyses cells prior to performing the PCR.

Such agents are well known to the skilled artisan. The reaction productsare then analyzed, e.g., via gel electrophoresis. An increase in BVDVsurrogate marker mRNA levels relative to levels obtained from anon-infected bovine is indicative of BVDV in the animal being tested.Alternatively, an increase in BVDV surrogate markers in Al animalsrelative to Pt animals, or in PI animals, relative to Al animals, candifferentially diagnose acute infection, or persistent infection.

In an alternative method, a skin tissue sample is obtained from thebovine suspected of having acute or persistent BVDV infection. The cellsare then lysed and PCR performed. As above, an increase in BVDVsurrogate marker mRNA expression levels relative to those observed in anon-BVDV infected animal being indicative of BVDV in the test animal.

It is also possible to detect BVDV using immunoassays. In an exemplarymethod, blood is obtained from a bovine suspected of being infected withBVDV. As above, the blood may optionally be centrifuged through aHypaque gradient to obtain a buffy coat. The blood or buffy coat sampleis diluted and at least one antibody immunologically specific for BVDVsurrogate markers is added to the sample. In a preferred embodiment, theantibody is operably linked to a detectable label. Also as describedabove, the cells may optionally be lysed prior to contacting the samplewith the antibodies immunologically specific for BVDV surrogate markers.

Increased production of BVDV surrogate markers is assessed as a fuactionof an increase in the detectable label relative to that obtained inparallel assays using blood from non-BVDV infected cow. In yet anotherembodiment, the blood or buffy coat preparation is serially diluted andaliquots added to a solid support.

Suitable solid supports include multi-well culture dishes, blots, filterpaper, and cartridges. The solid support is then contacted with thedetectably labeled antibody and the amount of BVDV surrogate markerprotein (e.g., a protein or peptide encoded by a nucleic acid of Tables1-9) in the animal suspected of being infected with BVDV is comparedwith the amount obtained from a non-AI or PI animal as a function ofdetectably labeled antibody binding. An alteration in the BVDV surrogatemarker protein level in the test animal relative to the non-AI or PIinfected control animal is indicative of acute or persistent BVDV.

In another embodiment, a first antibody which binds to a first epitopeon a target protein is placed in the well of a cartridge. Whole blood,blood collected in the presence of anticoagulants (e.g., sodium citrate,heparin), plasma, or serum is placed into the well of the cartridge. Thetarget protein, if present in the sample, is bound by the firstantibody, and then migrates laterally by a wicking action, through afilter which has been sprayed with second antibody. The second antibodyhas affinity for a second epitope on the target protein, oralternatively for the first antibody. The second antibody is optionallylabeled with a detectable label (e. g. radiolabel, gold, biotin, enzyme,etc.) The second antibody localizes the antigen, and results in theappearance of a line on the filter. The first and second antibodies maybe generated against the full length target protein, or against theN-terminal or C-terminal halves of the target protein, so that theyrecognize different epitopes of the target protein.

The foregoing immunoassay methods may also be applied to any type ofsample, including a urine sample.

VII. Kits and Articles of Manufacture

Any of the aforementioned products or methods can be incorporated into akit which may contain a BVDV specific polynucleotide, anoligonucleotide, a polypeptide, a peptide, a solid support (e.g.,filters, cartridges, gene chips) an antibody, a label, marker, orreporter, a pharmaceutically acceptable carrier, a physiologicallyacceptable carrier, instructions for use, negative and positive controlsamples, a container, a vessel for administration, an assay substrate,or any combination thereof.

The following materials and methods are provided to facilitate thepractice of the invention. The Examples illustrate certain embodimentsof the invention. They are not intended to limit its scope in any way.

EXAMPLE 1

Bovine viral diarrhea virus (BVDV) infections are responsible forimportant economic losses due to reproductive wastage, and respiratoryand digestive disease in cattle. Infection of the early developing fetusfrequently results in persistent infection (Pt). PI animals are the mainsource of new infection in herd mates. The complex host-viralinteractions resulting from persistent infection are minimallyunderstood, particularly in the bovine host. We hypothesized that geneexpression would differ in bloods collected from PI when compared tonon-infected steers. In preliminary studies, bloods were collected fromthree PI or three control steers and were processed to yield totalcellular RNA. Labeled RNA was used to screen six independent bovineAffymetrix DNA chips, and analyzed for fold-changes at the University ofColorado Health Sciences Center Microarray Facility. The top 100up-regulated genes belonged to MHC class I (45-fold), antiviral (32-100fold), transcription factor (8-12 fold), interferon stimulated genes(5-28 fold), bone remodeling (4-9 fold), and chemokine (2-4) families.The top 100 down-regulated genes belonged to adhesion (5-10 fold),T-cell receptor (5-10 fold), extracellular matrix (3-5 fold), growthfactor (2-3 fold), and transcription factor (2-3 fold) families. Weconclude from these findings that persistent infection with BVDV resultsin antiviral responses in blood cells which includes induction of type 1interferon-induced genes, chemokine-mediated immune responses and boneremodeling with a concomitant suppression of extracellular remodeling,adhesion and T-cell-mediated responses. Thus, in accordance with thepresent invention, single and/or multiplexing diagnostics foridentifying cattle that are persistently infected with BVDV areprovided.

BVDV is divided into two biotypes: cytopathic (cp) and non-cytopathic(nep). There are at least two recognized genotypes based on the sequenceof the 5′ untranslated region, type I and type II, and additionalsub-genotypes. Within each genotype, there are several strains thatcause different degrees of clinical signs. The pathogenesis of BVDVinfection has features that are unique to this virus and that vary withthe virulence of the viral strain and age of the animal at the time ofinfection.

Particularly interesting are the outcomes of infection of the fetus.Infection of the fetus during the first 150 days of gestation, when theimmune system has not yet developed, can lead to the generation of PIcalves (FIG. 1). Some of these PI calves die soon after birth, butothers live for relatively long periods of time without showing anyclinical signs. PI animals cannot eliminate the infecting BVDV fromtheir system and continuously release high amounts of virus in theirbodily secretions. This makes them a continuous source of infectionwithin the herd and potentially to other herds. When the infectionoccurs after 150 days of gestation (post-development of the immunesystem) or after birth, the infection is referred to as acute and theseanimals, which clear the virus, are frequently called transientlyinfected (TI). The clinical manifestations of acute infections with BVDVrange from sub-clinical, or unapparent infections, to embryonic death,abortions, stillbirths, and malformed or slow-growing calves. Althoughrecent studies indicate that transient infection of the fetus may causelong-term detrimental effects in the development of the calf, the basisfor these effects is not clearly understood.

Acute infection with the nep biotype results in inhibition ofdouble-stranded RNA-induced apoptosis and type 1 interferon, bothindicated as plausible contributors to the establishment of persistentinfection. However, in vitro and in vivo infections with the cp biotypeinduce apoptosis and are associated with increased type I interferonproduction.

The role of PI animals in the perpetuation of BVDV infections cannot beoveremphasized. The presence of a single PI calf in a herd can causesevere losses within that herd and any herd with which the PI calf makescontact. The large percentage of seropositive cattle due to vaccinationin the USA diminishes the value of serology as a monitoring test. Inaddition, PI animals have absent, extremely low or fluctuating titersagainst the strain that causes the persistent infection, potentiallyleading to misdiagnosis, particularly when serology is used as the onlytest. Current diagnostic tests based on the detection of viral antigenor viral RNA are effective tools for the identification of postnataldetection of PI animals. However, since PI animals shed high amounts ofvirus, early elimination of PI fetuses should greatly contribute todisrupting the infectious viral cycle.

In other preliminary studies, we demonstrated that calves persistentlyinfected with BVDV present a differential pattern of gene expression inthe blood when compared to vaccinated or acutely infected age-matchedherd mates. We investigated the gene expression profiles in the wholeblood of one year old PI and non-infected calves that were naturallyinfected with BVDV using DNA microarray approaches.

Blood samples were collected from three BVDV PI and three vaccinated,non-infected control calves in sodium citrate tubes and placed on ice.RNA was isolated using the QIAamp RNA Blood Mini Kit following themanufacturer's instructions (Qiagen). Red blood cells were selectivelylysed, and white cells were collected by centrifugation. White cellswere then lysed using highly denaturing conditions, which immediatelyinactivate RNases. After homogenization using the QIAshredder spincolumn, the sample was applied to the QIAamp spin column. Total RNAbinds to the QIAamp membrane and contaminants were washed away, leavingpure RNA which was eluted in 30-100 μl RNase-free water.

The total RNA was isolated and used for transcriptional profiling byscreening the Affymetrix bovine DNA chip. Biotinylated cRNA (15 μg),generated from each RNA sample (n=6 total), was hybridized to the bovineAffymetrix GeneChip (features of which are shown below) Array (n=6total). Data were analyzed using Affymetrix Microarray Suite Softwareversion 5.0 for absolute and pair-wise comparison analyses. Normalizedexpression values for the mean and standard deviation of three replicateaverage difference scores were calculated for each gene. Comparisonswere performed using the Student's t test (P<0.05 was consideredsignificant). The raw data were interpreted using GeneSpring (version5.0, Silicon Genetics, Redwood, Calif.) and GeneSifter software (vizXLabs, LLC, Seattle, Wash.) Critical Specifications Bos taurus (Bovine)probe sets 24,072 Bos taurus (Bovine) transcripts approximately 23,000UniGene clusters approximately 19,000 Unique probe sets to singlespecies: Number of arrays in set one Array format 100 Feature size 11 μmOligonucleotide probe length 25-mer Probe pairs/sequence 11Hybridization controls: bioB, bioC, bioD, from E. coli and cre from P1B. subtilis Poly-A controls: dap, lys, phe, thr, trp from B. subtilisHousekeeping/Control genes: actin, GAPDH, eflα, 5.8S rRNA, 12S rRNA, 18SrRNA, cyclophilin B, glutathione S-transferase, lactophorin, translationinitiation factor elF-4E Detection sensitivity 1:100,000¹¹As measured by detection in comparative analysis between a complextarget containing spiked control transcriptions and a complex targetwith no spikes

Results

Two hundred genes were up-regulated in blood from PI when compared tovaccinated control calves. Known attributes of the top 100 up-regulatedgenes and fold changes are listed below. 45 fold MHC Class I Molecules10-32 fold Antiviral genes 8-12 fold Signal Transduction Molecules 5-28fold Type 1 Interferon-Induced Genes (FIG. 2) 4-9 fold Bone RemodelingGenes 3-6 fold Cytoskeletal Remodeling Genes 2-4 fold Chemokine Ligandsand Receptors

One hundred genes were down-regulated in blood from PI when compared tocontrol calves. Known attributes of the top 100 down-regulated genes andfold changes are listed below. 5-10 fold Adhesion Molecules 5-10 fold TCell Receptors 3-5 fold Extracellular Matrix 2-3 fold Growth Factors 2-3fold Chemokine Ligands 2-3 fold Transcription FactorsSee FIG. 2.

Following more extensive and stringent analysis of microarray datadescribed above, the following up-regulated (Table 1) and down-regulated(Table 2) genes were identified as preferred bovine blood markers forBVDV persistent infection (n =2 steers) when compared with controls (n=3steers). MX2, 2′-5′ oligoadenylate synthetase (OAS) andInterferon-Stimulated Protein, 15 kDa serve as examples of good markersfor cattle with a BVDV PI infection when compared to non-infectedcontrols. See FIG. 3.

As can be seen by the data presented herein, persistent infection withBVDV causes up-regulation and down-regulation of genes in blood cells.Persistent infection with BVDV results in antiviral responses in bloodcells. The results show induction of interferon-induced genes,chemokine-mediated immune responses and bone remodeling genes.Suppression of extracellular remodeling, adhesion and T-cell-mediatedresponses is also observed. One gene, called interferon stimulated gene15 or ISG15 was confirmed to be up-regulated in bloods from PI whencompared to control vaccinated steers using blood cell mRNA and RealTime PCR approaches (FIGS. 2 and 3).

EXAMPLE 2

We hypothesized that gene expression in white blood cells would differin pregnant heifers carrying PI, TI or uninfected (control) fetuses.Non-vaccinated heifers were purchased, confirmed to be seronegative forBVDV and were placed on growing rations until they were old enough to beartificially inseminated and confirmed to be pregnant. Heifers wereinfected with noncytopathic BVDV2 on day 75 to generate PI fetuses, onday 175 to generate TI fetuses, or were not infected (n=6 heifers pertreatment). Bloods were collected on days 0, 37, 75, 78, 82, 90, 120,160, 175, 178, 182 and 190 of gestation for RNA, serology and virology.Fetuses were delivered on d. 190 by C-section and necropsied. Maternalblood mRNA on day 190 of gestation was screened using the bovineAffymetrix gene chips.

BVDV infection in heifers and fetuses was confirmed using ELISA andqRT-PCR. Infected pregnant heifers were seropositive for BVDV by days15-45 post infection. PI fetuses weighed less and were smaller withmaldeveloped bone and muscle tissue (P<0.05) when compared to TI or UIfetuses. Screening of 24,000 transcripts on the bovine Affymetrix DNAchip using mRNA from blood cells of heifers on day 190 of pregnancyrevealed 67 differentially expressed genes (1.5 fold or greater; P<0.05)based on infection status of the fetus: 32 genes in PI vs. TI, 26 genesin PI vs. control and 47 genes in TI vs. control. These genes wereclassified based on ontology analysis in primary categories of immuneresponse, antigen presentation, inflammatory response, chemotaxis,protein folding and modification, transport, and defense response tobacteria. Specific genes that are differentially expressed are describedin Tables 3-9.

TABLE 9 Analysis of blood gene expression using a three-way ANOVA inheifers carrying control vs. TI vs. PI fetuses. Analysis represents thetop 24 genes that were differentially expressed in at least one of thetreatment groups. The value in the ANOVA column represents the P valuewhen determining if one of the three means differs. Please refer to theheat plot in FIG. 4 for an illustration of these data. Control TI PIControl PI TI Gene ANOVA Mean Mean Mean SEM 1 SEM 2 SEM 3 IdentifierGene Title 0.006032 8.00309 10.9193 8.96783 0.188264 0.138741 0.660686CK960499 2′-5′-oligoadenylate synthetase 1 (OAS1) 0.003154 10.11511.9521 10.7336 0.256113 0.176554 0.231053 NM_174366interferon-stimulated protein. 15 kDa 0.003028 6.90223 8.43096 7.265290.101244 0.219103 0.222812 CK955157 XP_513514.1 similar toInterferon-induced protein 44 (Antigen p44) 0.003101 8.16371 9.688178.57334 0.191589 0.15614 0.212549 CB460780 XP_513514.1 similar toInterferon-induced protein 44 (Antigen p44) 0.003419 6.74951 8.135737.04487 0.104431 0.170809 0.23341 CK777675 XP_513514.1 similar toInterferon-induced protein 44 (Antigen p44) 0.001684 7.22418 8.766547.80757 0.137263 0.093874 0.232921 CB433489 pir: S48218 (H. sapiens)S48218 microtubular aggregate protein - hu

0.008299 8.21365 9.31672 8.57504 0.110353 0.233574 0.116391 CB432365XP_520524.1 similar to DEAD/H (Asp-Glu-Ala-Asp/His) box polype

0.00315 8.16901 9.22019 9.12652 0.048718 0.109524 0.209197 CB445920Transcribed sequences 0.002321 9.87333 9.15127 9.88773 0.12978 0.0954430.033274 CK953227 XP_518477.1 similar to beta-tubulin cofactor C [Pantroglodytes] 0.009597 5.57083 5.66441 6.21629 0.161879 0.077099 0.026107CK971667 XP_524698.1 similar to TAL1 (SCL) interrupting locus; SCLinterrup

0.000202 8.26679 7.64432 8.09498 0.036558 0.065628 0.028333 BP103941XP_521146.1 similar to ATRX [Pan troglodytes] 0.005323 6.91205 6.142057.06362 0.125015 0.061868 0.179441 CB534327 Component 3 0.003199 5.258865.54151 5.89927 0.099747 0.072327 0.051183 CB443429 NP_001804.1centromere protein E, 312 kDa [Homo sapiens] 0.00661 7.76552 7.114998.06201 0.028002 0.114012 0.201076 CB460964 ATP-binding cassettetransporter subfamily B, member 1 (ABCB1) 0.006969 5.88815 6.501566.25403 0.108627 0.090517 0.04993 CK770915 XP_218427.2 similar topoliovirus receptor homolog [Rattus norvegi

0.002739 4.50737 4.44439 5.47889 0.218718 0.048776 0.067073 CB461169Transcribed sequences 0.003618 7.02454 6.11085 7.19863 0.035911 0.2345150.075039 M37974 osteoglycin (osteoinductive factor, mimecan) 0.0072587.29919 7.80462 7.90756 0.145198 0.007311 0.065739 CK950633 similar toNP_612565.1 kinesin family member 23 [Homo sapiens] 0.006348 9.1115310.0777 9.28636 0.01768 0.189202 0.15531 CK848208 similar to XP_524747.1similar to histocompatibility 28 [Pan troglod

0.000656 4.52089 5.16352 5.09881 0.106526 0.017049 0.016545 CB168658Transcribed locus 0.0004 5.60571 6.28826 6.3913 0.053773 0.0634460.087055 CB463330 Succinate semialdehyde dehydrogenase (NAD(+)-dependentsuccir

0.002006 4.94446 3.99497 3.79603 0.173649 0.094759 0.123417 CB534503Transcribed locus, weakly similar to XP_519213.1 paraoxonase 2 [

0.009713 10.8281 9.92825 9.33185 0.253205 0.201907 0.221374 NM_175827chemokine (C—C motif) ligand 5 0.00064 11.0449 7.57803 10.8921 0.2580420.509224 0.189688 AB008573 MHC class I heavy chain, partial cds, cloneP5647.6m

Early detection of persistently infected calves is the best method ofcontrolling and eradicating BVDV from infected herds. Current diagnostictests for BVDV include skin biopsies and serology which do notdistinguish acutely infected animals from persistently animals. These PIanimals go undetected and continue to propagate virus within the herd.

Current methods of detection also have a high rate of false negativesdue to the fact that they are based on mutating viral antigens. Mostimportantly, current tests cannot detect a pregnant cow/heifer which iscarrying a persistently infected calf. The present invention describes amethod whereby BVDV can be detected in acutely infected calves,persistently infected calves as well as cows or heifers carrying aninfected fetus through the identification of differentially expressedsurrogate markers described in Tables 1-9. Surrogate markers can existas mRNA or protein antigens and allow differentiation of type of BVDVinfection. By the methods listed previously persistently infected (PI)calves can be differentiated from acutely infected calves and acutelyinfected calves can be distinguished from non-infected calves. Heifersand cows carrying PI fetuses can also be distinguished from heifers/cowscarrying transiently infected fetuses and cows/heifers carrying PI or TIfetuses can be distinguished from cows/heifers carrying non-infected(normal) calves. The same markers can be used to create therapeutics fortreating secondary effects of viral disease and monitoring theanti-viral response in infected cattle. The global approach presentedherein will aid the prevention, diagnosis, control and treatment ofcattle infected with BVDV as well as other RNA viruses.

While certain of the preferred embodiments of the present invention havebeen described and specifically exemplified above, it is not intendedthat the invention be limited to such embodiments. Various modificationsmay be made thereto without departing from the scope and spirit of thepresent invention, as set forth in the following claims.

1. A method for diagnosing BVDV in a ruminant test animal comprising: a)obtaining a biological sample from a test animal and from a non-BVDVinfected animal; b) contacting said sample with an agent having affinityfor at least one differentially expressed BVDV surrogate marker shown inTables 1-9; and d) diagnosing the presence of BVDV via detection of atleast one differentially expressed BVDV surrogate marker, an alterationin the expression level of said BVDV surrogate marker obtained from saidtest animal, relative to that obtained from said non-BVDV infectedanimal being indicative of BVDV in said test animal.
 2. The method ofclaim 1, wherein said at least one BVDV surrogate marker is a nucleicacid and said expression level is up-regulated in response to BVDVinfection.
 3. The method of claim 1, wherein said at least one BVDVsurrogate marker is a nucleic acid and said expression level isdown-regulated in response to BVDV infection.
 4. The method of claim 1,wherein said at least one BVDV surrogate marker is a nucleic acidimmobilized on a gene chip.
 5. The method of claim 1, wherein saidbiological sample is selected from the group consisting of blood, urine,skin, serum, milk, sputumr, saliva, and tears.
 6. The method of claim 1wherein said biological sample comprises blood cells or skin cells whichare lysed to release nucleic acids therein.
 7. The method of claim 6,wherein said released nucleic acids are amplified.
 8. The method ofclaim 1, wherein said ruminant animal is a persistently infected steeror heifer and at least one BVDV surrogate marker is a nucleic acid whichis upregulated and is selected from the group consisting of CK945739,CK777968,NM_(—)173941 and CK960499. which is upregulated and is selectedfrom the group consisting of CK945739, CK777968, NM_(—)173941 andCK960499.
 9. The method of claim 1, wherein said ruminant animal is apersistently infected steer and at least one BVDV surrogate marker is anucleic acid which is downregulated and is selected from the groupconsisting of CK848330, BP 102272, AU278490 and BE723387.
 10. The methodof claim 1, wherein said ruminant animal is a heifer carrying apersistently infected fetus and at least one BVDV surrogate marker is anucleic acid which is upregulated and is selected from the groupconsisting of CK977019, CA923353, CB461169, and CB445920.
 11. The methodof claim 1, wherein said ruminant animal is a heifer carrying apersistently infected fetus and at least one BVDV surrogate marker is anucleic acid which is down regulated and is selected from the groupconsisting of NM_(—)175827, CB425639, NM_(—)174511 and CB534503.
 12. Themethod of claim 1, wherein said ruminant animal is a heifer carrying atransiently infected fetus and at least one BVDV surrogate marker is anucleic acid which is upregulated and is selected from the groupconsisting of CK960499, CB464371, NM_(—)174366, and CB433489.
 13. Themethod of claim 1, wherein said ruminant animal is a heifer carrying atransiently infected fetus and at least one BVDV surrogate marker is anucleic acid which is down regulated and is selected from the groupconsisting of CB444277, AB008573, CB433789 and AV609250.
 14. A gene chipfor performing the method of claim 1 comprising a plurality of BVDVsurrogate markers selected from the group consisting of CK945739,CK777968, NM_(—)173941, CK960499, CK848330, BP102272, AU278490,BE1723387, CK977019, CA923353, CB461169, CB445920, NM_(—)175827,CB425639, NM_(—)174511, CB534503. CK960499, CB464371, NM_(—)174366,CB433489, CB444277, AB008573, CB433789 and AV609250.
 15. The method ofclaim 1, wherein said at least one BVDV surrogate marker is apolypeptide and said expression level is up-regulated in response toBVDV infection.
 16. The method of claim 1, wherein said at least oneBVDV surrogate marker is a polypeptide and said expression level isdown-regulated in response to BVDV infection.
 17. The method of claim 1,wherein said at least one BVDV surrogate marker is a polypeptide orfragment thereof immobilized on a solid support.
 18. The method of claim1, wherein said ruminant animal is a persistently infected steer and atleast one BVDV surrogate marker is a polypeptide or fragments thereofwhich is upregulated and is selected from the group consisting ofpolypeptides thereof encoded by CK945739, CK777968, NM_(—)173941 andCK960499.
 19. The method of claim 1, wherein said ruminant animal is apersistently infected steer and at least one BVDV surrogate marker is apolypeptide or fragments thereof which is downregulated and is selectedfrom the group consisting of polypeptides encoded by CK848330, BP102272,AU278490 and BE723387.
 20. The method of claim 1, wherein said ruminantanimal is a heifer carrying a persistently infected fetus and at leastone BVDV surrogate marker is a polypeptide or fragment thereof which isupregulated and is selected from the group consisting of polypeptidesencoded by CK977019, CA923353, CB461169, and CB445920.
 21. The method ofclaim 1, wherein said ruminant animal is a heifer carrying apersistently infected fetus and at least one BVDV surrogate marker is apolypeptide or fragment thereof which is down regulated and is selectedfrom the group consisting of polypeptides encoded by NM_(—)175827,CB425639, NM_(—)174511 and CB534503.
 22. The method of claim 1, whereinsaid ruminant animal is a heifer carrying a transiently infected fetusand at least one BVDV surrogate marker is a polypeptide or fragmentthereof which is upregulated and is selected from the group consistingof polypeptides encoded by CK960499, CB464371, NM_(—)174366, andCB433489.
 23. The method of claim 1, wherein said ruminant animal is aheifer carrying a transiently infected fetus and at least one BVDVsurrogate marker is a polypeptide or fragment thereof which is downregulated and is selected from the group consisting of polypeptidesencoded by CB444277, AB008573, CB433789 and AV609250.
 24. A solidsupport for performing the method of claim 1 comprising a plurality ofBVDV surrogate polypeptide markers selected from the group consisting ofpolypeptides encoded by CK945739, CK777968, NM_(—)173941, CK960499,CK848330, BP102272, AU278490, BE723387, CK977019, CA923353, CB461169,CB445920, NM_(—)175827, CB425639, NM_(—)174511, CB534503. CK960499,CB464371, NM_(—)174366, CB433489, CB444277, AB008573, CB433789 andAV609250.
 25. The method of claim 1, wherein said ruminant test animalis selected from the group consisting of a bovine a steer, a pregnantbovine, a bovine fetus and a bovine calf.
 26. The method of claim 1,wherein said agent having affinity for said BVDV surrogate marker isselected from the group consisting of at least one nucleic acid whichspecifically hybridizes with at least one nucleic acid shown in Tables1-9, and an antibody immunologically specific for at least onepolypeptide encoded by a nucleic acid shown in Tables 1-9.
 27. Themethod of claim 26, wherein said agent is detectably labeled.
 28. A kitfor differentially diagnosing BVDV infection via the method of claim 1,comprising at least one BVDV surrogate marker detector molecule, andreagents for detection of the same said optionally comprising a genechip comprising a plurality of BVDV surrogate markers selected from thegroup consisting of CK945739, CK777968, NM_173941, CK960499, CK848330,BP102272, AU278490, BE723387, CK977019, CA923353, CB461169, CR445920,NM_(—)175827, CB425639, NM_(—)174511, CB534503. CK960499, CB464371,NM_(—)174366, CB433489, CB444277, AB008573, CB433789 and AV609250 or asolid support comprising a plurality of BVDV surrogate polypeptidemarkers selected from the group consisting of polypeptides encoded byCK945739, CK777968, NM_(—)173941, CK960499, CK848330, BP102272,AU278490, BE723387, CK977019, CA923353, CB461169, CB445920,NM_(—)175827, CB425639, NM_(—)174511, CB534503. CK960499, CB464371,NM_(—)174366, CB433489, CB444277, AB008573, CB433789 and AV609250. 29.The kit of claim 28, wherein said BVDV surrogate marker detectormolecule is selected from the group consisting of a probe or primerwhich specifically hybridizes with a BVDV surrogate marker nucleic acid,and an antibody which specifically binds to a BVDV surrogate markerpolypeptide.
 30. A method as claimed in claim 1 further comprisingdetection of markers associated with transient or acute BVDV infection.31. A method for identifying agents useful for the treatment of viralinfections, comprising: a) providing a host cell expressing at leastBVDV surrogate marker in Tables 1-9 which is differentially expressed inresponse to viral infection and exposing said cell to an RNA virus underconditions suitable for infection to occur; b) incubating said hostcells in the presence and absence of said agent; and c) identifyingthose agents which modulate the expression of at least one BVDVsurrogate marker in treated cells when compared to untreated cells. 32.A method for detecting viral surrogate marker molecules in a test animalcomprising: a) obtaining a plurality of biological samples from saidtest animal and from a non-virally infected animal; b) contacting saidbiological sample with a composition comprising one or more viralsurrogate marker molecule detection reagents in an amount effective topermit detection and quantitation of a viral surrogate molecule, ifpresent, in said sample; and c) determining from b) the amount of saidviral surrogate marker molecule, wherein an alteration of levels of saidviral surrogate marker molecule, relative to those obtained fromnon-virally infected animals, is indicative of viral infection in saidtest animal.
 33. The method of claim 32, wherein a lack of alteration oflevels of said viral surrogate marker molecule indicates that the testanimal is not virally infected.
 34. The method of claim 32, wherein saidviral surrogate marker molecule is increased in a test subject infectedwith a virus selected from the group consisting of BVDV, Influenza, HIV,Ebola virus, FcLv, FIP virus, Bluetongue virus, West Nile Virus,hepatitis C Virus and Epizootic Hemorrhagic Disease Virus.